Mastitis is a highly infectious disease prevalent in dairy cattle and it is majorly caused by Escherichia coli (E. coli). The objective of present study is to investigate the occurrence of virulence genes, antimicrobial susceptibility and comparative analysis of E. coli (IVRI KOL CP4 and CM IVRI KOL-1) isolates from mastitis infected animal. Whole-genome sequencing (WGS) was performed using a PacBio RS II system and de novo assembled using Hierarchical Genome Assembly Process (HGAP3). Bacterial Pan Genome Analysis Pipeline (BPGA) was used for pangenome analysis. A set of 50 E. coli isolates were used for comparative analysis (48 collected from the database and 2 reference sequences). Core genes were further concatenated for phylogenetic analyses. In silico analysis was performed for antibiotic resistance and virulence gene identification. Both of the E. coli isolates carried many resistance genes including, b-lactamase, quinolones, rifampicin, macrolide, aminoglycoside and phenicols resistance. We detected 39 virulence genes in IVRI KOL CP4 and 52 in CM IVRI KOL-1 which include toxins, adhesions, invasins, secretion machineries or iron acquisition system. High prevalence of mastitis strains belongs to phylogroups A, although few isolates were also assigned to phylogenetic groups B1 and B2. In conclusion, the present study reported the presence of genes involved in Adherence, Iron acquisition, secretion system and toxins which shown to be crucial in MPEC pathogenicity. This is the first whole genome analysis of MPEC strains to be carried out in Indian isolate to highlights the spread of resistance and virulence genes in food animals.